• Clinical and Experimental Reproductive Medicine
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• v.34 no.2
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• pp.117-124
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• 2007

Objective: The aim of this study was to compare the efficacy of slow freezing with vitrification method for cryopreservation of mouse pronuclear stage embryos. Methods: Mouse pronuclear embryos obtained from superovulated mice and classified into 2 groups of slow freezing and vitrification. Slow freezing solution consisted of 1.5 M PROH, 0.1 M sucrose, while vitrification solution consisted of 40% ethylene glycol, 18% Ficoll and 0.5 M sucrose diluted in Dulbecco's phosphate-buffered saline supplemented with 10% SSS. Recovery and survival rates after thawing and development rates to hatching balstocyst stage were compared between two groups. Results: After freezing and thawing, recovery rate of slow freezing group was 93.8%, whereas vitrification group was 66.5% (p<0.01). Survival rate of recovered embryos were similar between two groups as 83.2% in slow freezing and 87.6% in vitrification. Embryo development rates to 2-cell stage after 24 hrs (77.0% vs 59.1%), 4-cell after 48 hrs (72.6% vs 53.3%), blastocyst after 96 hrs (53.1% vs 40.1%) of thawing were significantly higher in vitrification group than those of slow freezing group, respectively. Conclusion: The vitrification method may provide better developmental competence of frozen-thawed embryos than that of slow freezing method for cryopreservation of mouse pronuclear stage embryos.

• Development and Reproduction
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• v.13 no.4
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• pp.353-362
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• 2009

GnRH and its receptor are known to express locally in the ovary and to regulate the ovarian function by affecting on granulosa and lutein cells. It has been reported that GnRH directly causes apoptosis in the granulosa and lutein cells of the ovary. However, whether the apoptosis of the cells by GnRH is recovered by FSH as an anti-apoptotic factor is not yet known. In this study, we evaluated the apoptosis and the production of progesterone $(P_4)$ and estradiol $(E_2)$ after treatment with 5, 50, and 100 ng/$m\ell$ GnRH and 1 IU/ml FSH in the granulosa-lutein cells that are obtained during oocyte-retrieval for IVF-ET. Results of DNA fragment analysis and TUNEL assay demonstrated that DNA fragmentation and the rate of apoptotic cells were increased in a dose-dependent manner showing a significant increase in the cells treated with 100 ng/$m\ell$ GnRH. In addition, we found that FSH suppresses the apoptosis of the cells induced by GnRH. In the results of chemiluminescence assay for $P_4$ and $E_2$, $P_4$ production was decreased by GnRH treatment, whereas $E_2$ production was not changed. We also demonstrated that FSH inhibits the suppressive effect of GnRH on $P_4$ production as the result of apoptosis. The present results suggest that GnRH agonist using in ovarian hyperstimulation protocol might induce the dysfunction of the ovary, but its function could be recovered by FSH. These results also will be expected to use as the basic data to elucidate the physiological role of GnRH and to develop new ovarian hyperstimulation protocols for IVF-ET.

• Korean Journal of Animal Reproduction
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• v.25 no.2
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• pp.163-169
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• 2001

OSBA(oocytes-sperm binding assay) is a tool developed for rapid test of optimal condition of IVF medium and protein source by binding ability of mouse sperm and egg. Mouse oocyte-cumulus complexes were prepared by removing of the cumulus cells with 0.1% hyaluronidase. 10$\pm$2 oocytes per 30 ${mu}ell$ medium drop were inseminated with 3 ${mu}ell$ sperm suspension and were cultured f3r 3 hours and 24 hours, respectively. And the oocytes were recovered gently and the No. of sperm bound on oocytes were counted. In the Exp. 1, the ratio of oocytes bound with one sperm at least were 60.2%(50/83), 2%(2/77) and 100%(79/79) in the medium with no protein, FBS(15%, v/v) and BSA(0.4%. w/v), respectively, Fetal bovine serum(FBS) seriously inhibited sperm binding on oocyte, although bovine serum albumin(BSA) promoted the binding ability. The inhibiting effect of FBS was dependent on the concentration of FBS. The sperm binding ability according to oocyte maturity was tested in the Exp. 2. There was no significant difference between Met. II (mature) and Met. I (intermediate mature) oocytes in the number of oocytes bound with sperm and the number of sperm bound on oocytes. Finally, in Exp. 3, two batches of Ham's F10 medium with good and poor quality by OSBA were tested (The ratios of embryos developed from PN 1-cell stage to hatched blastocyst; 25% vs. 70%). In the medium with good quality, sperm binding ability was significantly increased (P ＜ 0.05). The ratio of oocytes bound with one sperm at least was 66% and 90% in the medium with poor and good quality, respectively. Conclusively, It was possible to test IVF medium condition rapidly and easily by OSBA.

• Korean Journal of Animal Reproduction
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• v.26 no.2
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• pp.193-199
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• 2002

This study was conducted to investigate the optimal physiologic mating time in Hanwoo for protection to decrease of reproductivity and improvement of production of offspring. We observed 32 cows that were devide into 4 parts of treatment : T1(12 months of age and 0.5kg daily gain), T2(12 months of age and 0.8kg daily gain), T3(15 months of age and 0.5kg daily gain) and T4(18 months and 0.5kg daily gain). The first heat of treated cows was 263.3$\pm$6.4 days and average weight was 181.1$\pm$11.3kg. It was revealed the conception rates of first insemination were 25%(T1), 75%(T4) and number of insemination of T3 and T4(both 1.5) was lower than T1 and T2(2.3 and 2.4). In return of estrus after heifer's first parturition, they(T1, T2, T3 and T4) showed 66.2 days, 76.7 days, 62.4 days and 68.5 days respectively and the average was 65.7 days. Plasma progesterone(P4) concentration was nearly the same during the observation periods of treated cows and P4 was released just after 12 months. Only 5 cows (15.6%) in 32 were showed normal estrus cycle and ovulation before 12 months. Before and after parturition, P4 concentration was decreased fastly and then there was no detection of P4 from after parturition to 40 days after milking. P4 would be released again on 45 day after parturition. The results were summarized as that the optimal mating time of Hanwoo heifers was decided by the 14 months of age, 110 cm height and 265kg weight.

• Journal of Practical Agriculture & Fisheries Research
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• v.4 no.1
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• pp.110-118
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• 2002

This study was conducted to investigate the physiology of optimal reproductive age in Hanwoo for protection to decrease of reproductivity and improvement of production of offspring. Thirty two cows were devided into 4 groups of treatments : T1(12 months of age and 0.5kg daily gain), T2(12 months of age and 0.8kg daily gain), T3(15 months of age and 0.5kg daily gain) and T4(18 months of age and 0.5kg daily gain). The days of the first heat of treated cows were 263.3±6.4 days and average weight was 181.1±11.3kg. The conception rates of first insemination were 25%(T1) and 75%(T4), and the number of insemination of T3 and T4(both 1.5) was lower than those of T1 and T2(2.3 and 2.4), respectively. With regard to estrus return after the first parturition, T1, T2, T3 and T4 showed 66.2,76.7, 62.4 and 68.5 days, respectively, indicating the average days of estrus return was 65.7. Plasma progesterone(P4) concentration was nearly the same during the observation periods of treated cows and P4 began to be detected after 12months. Only 5(15.6%) out of 32cows showed normal estrus cycle and ovulation before 12 months. During the peri-parturition period, P4 concentration was rapidly decreased and there was no detection of P4 from parturition to 40 days after milking. P4 would be released again on 45 day after parturition. The results imply that the optimal reproductive age of Hanwoo heifers would be around at the 14 months of age, 110cm height and 265kg weight.

• Clinical and Experimental Reproductive Medicine
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• v.12 no.2
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• pp.25-37
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• 1985

Four points on the basal body temperature (B.B.T.) curve was correlated with the estimated time of ovulation, as determined by serial ultrasound in 50 induced menstrual cycles from 22 subjects. The time of ovulation was estimated by measuring the maximal diameter of follicles and observing the morphologic changes within the ovary from follicle to corpus luteum. The results were as following; 1. The diameter of the follicle measured at the day before disappearance was 21.1 mm on an average (S.D.: 2.14). The average follicular growth for 4 days before ovulation was measured at a rate of 2.8 mm/day, and rapid growth of follicle was observed 3.1 mm/day at the day before. 2. The changes associated with rupture of the follicles were the followings, in order of frequency; decrease in size(94%), disappearance of follicles(64%), fluid in the Cul-de-Sac(26%) and increased internal echoes(16%). 3. Only 20 of 50 cycles, exhibited a BBT dip and correlated with the estimated time of ovulation by ultrasound in 2 of which cases(10%). BBT nadir, 30 of 50 cycles, correlated in 5(16.7%). The first day of hyperthermic plateau(FDHP) and BBT coverline was exhibited in all cycles, correlated in 41(82%) and 35(70%) cases. 4. The relationship between the diameter of dominant dominant follicle, measured by ultrasound, and the basal body temperature curve were as following. During cycles in which dip was observed on the BBT curve, the follicular diameter were 10.5${\pm}$2.12 mm on 4 days prior to the point (D-4), and 12.5${\pm}$2.12 mm (D-3), 15.5${\pm$2.12 mm (D-2), 17.0${\pm}$1.41 mm (D-1) and 21.5${\pm}$2.12 mm just prior to the dip (D-0). In the nadir; 9.6${\pm}$1.67 mm (N-4), 12.8${\pm}$1.79 mm (N-3), 16.2${\pm}$1.92 mm (N-2), 18.2${\pm}$2.17 mm (N-1) and 21.4${\pm}$2.61 mm (N-0). In the First day of Hyperthemic Plateau (FDHP); 9.8${\pm}$1.36 mm (F-4), 12.4${\pm}$1.41 mm (F-3),15.1${\pm}$1.57 mm (F-2), 18.1${\pm}$1.67 mm (F-1) and 21.2${\pm}$2.25 mm (F-0). In the BBT coverline endopint; 9.9${\pm}$.39 mm (C-4), 12.5 ${\pm}$1.44 mm (C-3), 15.2${\pm}$1.64 mm (C-2), 18.0 ${\pm}$1.69 mm (C-1), and 21.2${\pm}$2.31 mm (C-0). 5. The relationship between the ultrasonographic signs of ovulation and the basal body temperature curve were as following. The BBT dip correlated with the ovulation in 2 cases, which revealed decrease in follicular diameter (100%), fluid pattem in the Cul-de-Sac (1 case, 50%) and complete disappearance of follicle (1 case, 50%). In the nadir (5 cases); the ultrasonographic signs of ovulation were decrease in follicular diameter (5 cases, 100%), fluid pattern in the Cul-de-Sac (1 case, 20%) and complete disappearance of follicle (3 cases, 60%). In the First day of Hyperthermic Plateau (41 cases); decrease in follicular diameter (40 cases, 97.6%), fluid pattern in the Cul-de-Sac (11 cases, 26.8%), appearance of internal echo and thickening of the wall (6 cases, 14.6%) and com plete disappearance of follicle (28 cases, 68.3%). In the BBT coverline endpoint (35 cases); decrease in follicular diameter (33 cases, 94.3%), fluid pattern in the Cul-de Sac (9 cases, 25.7%), appearance of internal echo and thickening of the wall (5 cases 14.3%) and complete disappearance of follicle (20 cases, 57.1%).

• Journal of Aquaculture
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• v.7 no.4
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• pp.189-205
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• 1994

Formerly, adult-tiger puffer, Takifugu rubripes with ova caught in the sea, were used for seedling production. But it was difficult to secure naturally-ripened adults. For the purpose of adult tiger puffer in captivity, this study was carried out. To determine the growth 220 tiger puffers hatched in 1990 (3-year-old) and 1991 (2-year-old) were used. For spawning and egg incubation leading to fry development, eggs were stripped from tiger puffers hatched in 1988 (5-year-old) and 1990 (3-year-old) through human chorionic gonadotropin (BCG) treatments. In May, 1993, mean body length and mean body weight of 2-year-old tiger puffer were $30.72\pm1.35cm\;and\;1,048\pm228 g,$ and that of 3-year-old tiger puffers were $36.02\pm1.17cm$ and $1,402\pm66g$ respectively. The relationship between body length (L) and body weight (W) of 2-year-old the tiger puffers during the experiment period was represented as $W\;=\;1.7892L^{31524}\times10^5$ (r= 0.9436) and that of 3-year-old, $W=\;3.2840L^{36099}\times10^6$ (r= 0.9070) respectively. The GSI in female 2-year-old-fish changed from $0.23\times0.l2\;to\;0.74\pm0.08$, during the experiment period, and in male it didn't change remarkably until November, but thereafter it increased and showed a peak of $8.69\pm5.09$. The GSI of 3-year-old-fish showed a peak of $8.05\pm5.58$ in April in female and $12.65\pm4.60$ in May in male. The change of HSI in 3-year-old-fish was correlative to the change of GSI, but in 2-year-old-fish it was little correlative. In female gonad of 2-year-old tiger puffer, the mature oocytes reached $350{\mu}m$ in April, but thereafter they didn't spawn and became atrophied. But in male gonad, a great number of spermatozoa were crowded in the testicular lobuli in April. Female gonad of 3-year-old tiger puffer had the mature oocytes of 650 pm in March and the ripe oocytes, $900{\mu}m$ in April. Male testis development was similar to that of 2-year-old-fish. Egg-stripping after hormone treatments was possible past 139 hours and 142 hours from each of two 5-year-old-fish (500IU/kg, BW), and after 114 hour from a 3-year-old-fish (1,000 IU/kg, BW) under water temperature $16.3\~17.8^{\circ}C$. Eggs stripped amounted was 650 g and 400 g from two 5-year-old-fish and 610 g from the 3-year-old-fish, and fertilization rates were $98.0\%,\;97.4\%\;and\;96.5\%$ respectively. All the hatched larvae devloped into normal fry.

• Development and Reproduction
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• v.6 no.2
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• pp.97-104
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• 2002

In most mammals, mature oocyte-cumulus complexer(OCCs) ovulate into the oviduct where fertilization by sperm takes place. However, the complex that fail to fertilize eventually undergoes degeneration while they reside in the oviduct. Yet there is no blown mechanism how both oocyte and cumulus cells degenerate. Using human follicular fluid (hFF), bovine oviductal tissue extract (BOX) and mouse OCC, the present study aimed to find how the oviduct influence the viability of the oocyte and cumulus cells in vitro. There was no difference of oocyte maturation rate between the control and BOX-treated groups. However, there was a significant difference in the survival of cumulus cells between two groups. Cumulus cells cultured in the presence of hFF alone underwent initially expansion and then they formed monolayer in the culture dish. Even after 72 hr, they proliferated well and showed fibroblast-like morphology. Cumulus cells cultured in the presence of both hFF and BOX also expanded after 24 hr, however, after 72 hr culture, they eventually detached and degenerated. Cumulus cells cultured in the BOX alone gave a similar drastic result. When the cumulus cells cultured in the presence of BOX were stained with DAPI, their nuclei showed partial condensation and fragmentation. After detailed analysis of these cells by TUNEL assay, many nuclei of them exhibited well stained spots indicating the signs of apoptosis. Based upon these observations, it is suggested that BOX might possess a factor that leads mouse cumulus cells to undergo apoptosis in vitro.

• Journal of Embryo Transfer
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• v.22 no.2
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• pp.89-95
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• 2007

The present study was conducted to examine some factors affecting in vitro development and fecundity of embryos recloned with somatic cell nuclear transfer (SCNT). Fibroblast cells retrieved from the ear of a 3-week-old, cloned Korean goat (Jinsoonny) were used as karyoplast donors and serum-starvation was conducted in tissue culture medium (TCM)-199 supplemented with 0.5% FBS. Recipient oocytes were surgically collected by flushing the oviducts 35 h after hCG injection following FSH priming. The zonae pellucidae of the oocytes were partially perforated with a laser drill and a donor cell was transferred into an enucleated oocyte. The couplets were electrically fused and activated by ionomycin (5 min) and 6-DMAP (4 h). The reconstructed embryos were cultured in mSOF medium containing 0.8% BSA at $39^{\circ}C$ in an atmosphere of 5% $CO_2$, 5% $%O_2$, 90% $N_2$ for 12 to 15 h. Re-cloned embryos (2- to 4-cell stages) were surgically transferred into the oviducts of the recipients and pregnancy was subsequently diagnosed by progesterone assay and ultrasound on Days 21 and 63 of pregnancy. The fusion rate following 1st fusion pulse was higher (p<0.05) in 2nd cloning (65.9%) compared to 1st cloning (51.0%), but it was not different in the other groups. The rate of cleavage after fusion was significantly higher (p<0.05) in 1st (77.7%) than in 2nd cloning (56.0%). A total of 175 re-cloned embryos were transferred into 28 recipients. On day 21 and 60 after transfer, 11 (39.3%) and 4 recipients (17.4%) were pregnancy, respectively. In comparison of pregnancy rate by estrous synchronization, a total of 66 and 109 re-cloned embryos were transferred into 11 recipients in natural estrus and 17 recipients in induced estrus, respectively. Five (45.4%) and 2 recipients (18.2%) in natural estrus were pregnant on days 21 and 63 while 6 (35.3%) and 2 (11.8%) recipients in induced estrus were pregnant, respectively. These results show that recloning of goat can be achieved by SCNT and estrous synchronization between donor and recipient animals may be one of the major factors affecting success rate.

• Development and Reproduction
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• v.12 no.1
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• pp.77-86
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• 2008

This study was carried out to investigate the effect of leukemia inhibitory factor (LIF) on the derivation of mouse ES cells from isolated blastomeres. Two-cell stage mouse embryos were obtained from superovulated BDF1 female mice. Collected embryos were cultured to blastocyst stage in culture medium supplemented with 0, 1,000, 2,500 or 5,000 U/mL of LIF. Cultured blastocysts were examined by counting the number of cells in the inner cell mass (ICM) and trophectoderm (TE) using differential staining method. When 2-cell embryos were cultured with 2,500 U/ml of LIF, the cell numbers of ICM significantly increased in comparing with those of the control($21.0{\pm}4.0$ vs. $15.9{\pm}5.0$, P<0.01) and 1,000 U/mL of LIF-containing group ($21.0{\pm}4.0$ vs. $16.6{\pm}4.9$, P<0.05). We used an ES cell establishment medium with 20% Knockout Serum Replacement and 0.01 mg/mL ACTH instead of fetal bovine serum. Establishing efficacy of ES cell lines were the highest in 2,500 U/mL of LIF-containing group as 36.7% (11/30). This culture medium was applied to the culture of isolated blastomeres and to derivate ES cell lines. Three ES cell lines (21.4%) from isolated blastomeres of 2-cell stage embryos were established. In further experiments, we could establish one ES cell line (4.0%) from single blastomere of 4-cell stage embryo. The subcultured ES cells and their embryoid bodies were characterized by analyzing gene expression for undifferentiation and differentiation marker gene using immunocytochemistry and RT-PCR. In conclusion, LIF supplementation in culture medium could increase the cell number in ICM of blastocysts and support derivation of ES cell lines from isolated blastomeres.